The present invention relates to fluorescent coumarin derivatives useful as instrument calibrators for certain homogeneous fluorescent immunoassay test procedures. In particular, the present compounds have advantageous water solubility and spectral characteristics as instrument calibrators for use in conjunction with certain homogeneous enzyme substrate-labeled fluorescent immunoassay methods.
In September 1979, the first product in the series of AMES TDA.TM. therapeutic drug assay test kits (Miles Laboratories, Inc., Elkhart, IN) was introduced, the AMES TDA Gentamicin test kit. The assay method used with the test kit is a homogeneous enzyme-substrate-labeled fluorescent immunoassay (SLFIA) technique; "homogeneous" because there is no separation step required in performing the assay, in contrast with "heterogeneous" techniques such as the radioimmunoassay (RIA). A key reagent in the test kit is a labeled conjugate comprising sisomicin (an aminoglycoside antibiotic which behaves immunochemically like gentamicin) covalently bound to a derivative of the fluorogenic enzyme substrate umbelliferyl-.beta.-D-galactoside. This Fluorogenic Gentamicin Reagent (FGR) is non-fluorescent under the conditions of the assay, however, hydrolysis catalyzed by .beta.-galactosidase yields a fluorescent product. When antibody to gentamicin binds FGR, it becomes virtually inactive as a substrate for .beta.-galactosidase.
Competitive binding reactions are established with a constant amount of FGR, a limiting amount of the antibody, and the test sample containing gentamicin: ##EQU1## The gentamicin in the test sample competes with FGR for binding to antibody. FGR not bound by antibody is hydrolyzed by .beta.-galactosidase to produce the fluorescent product. Hence the fluorescence produced is dependent on the gentamicin level in the sample. The fluorescence intensity is related to gentamicin level by means of a standard curve.
Fluorescence is measured on a fluorometer and recorded in arbitrary fluorescence units. In order that the instrument operator can properly set the instrument to the appropriate scale multiplier prior to reading the first actual assay result, the AMES TDA Gentamicin product includes a Range Adjustment Solution (RAS) comprising the fluorescent compound N-(phenyl)-7-hydroxycoumarin-3-carboxamide. The concentration of this fluorescent compound in the RAS is set such that the resulting fluorescence intensity of a prescribed diluted volume of the RAS under the conditions of the assay, i.e., room temperature, excitation wavelength of 400 nm and emission wavelength of 450 nm, approximates the fluorescence of the highest gentamicin level on the standard curve. Thus, the operator can preset the instrument to the maximum useable scale for actual assay runs. In addition, instrument function can be tested by assaying a series of dilutions of the RAS and checking the expected linearity of the relationship between observed fluorescence and RAS level.
Although the RAS compound, N-(phenyl)-7-hydroxycoumarin-3-carboxamide, is commercially available and has the desired spectral characteristics, its water solubility is quite poor for manufacturing purposes. Accordingly, it is the object of the present invention to provide compounds having the desired spectral characteristics for use in the Range Adjustment Solution together with increased water solubility, yet not involving a delicate or intricate synthesis.